This application relates to a sample measurement device and a method to detect extra cellular or intracellular ATP. The ATP is obtained from a culture medium or from a liquid, for example, a beverage, cosmetic, or pharmaceutical. The present invention measures growth of microorganisms, such as bacteria, yeast, mold, etc., using sequential measurements over time.
It is important in many industries, such as food preparation, medicine, beverages, toiletries, and pharmaceuticals, to provide clean and sanitary surfaces. In addition it is important to ensure that products are free of microorganisms such as bacteria which can contaminate and spoil the product. It is not enough to simply clean or sanitize a surface, or prepare a product under controlled sanitary conditions. Instead, a test must be performed to detect whether the surface or product is actually free of microorganisms. For instance, random areas of a surface, such as a food preparation surface, must be tested microorganisms to determine the general cleanliness of the surface. Or samples of the product itself must be tested to ensure that the product is not contaminated.
One of the oldest methods to check for presence of contaminants involves culturing samples for microorganisms. For example, a test surface is chosen and wiped with a swab, and then the swab is smeared onto a culture medium. Alternatively, a sample of the product itself, such as a beverage, cosmetic, or pharmaceutical, is placed in a culture medium. The medium is incubated and then checked for the presence of bacteria colonies grown in the medium. Over the years, various types of culture media have been developed along with numerous products based thereon. While the results of bacterial cultures are accurate, they are limited by the time it takes to incubate the culture, usually in the order of days.
In response for a need to obtain results more quickly, other methods for detecting microorganisms were developed. Research soon focused on the detection of biomass. Biomass includes living cells, dead cells, other biotic products such as blood, and food residue. It was discovered that biomass could be detected by detecting ATP, adenoisine triphosphate, a chemical found in all living organisms.
The specific test for ATP involves the "firefly" reaction. The following is the reaction: ##STR1## ATP, luciferin (D-luciferin cofactor), luciferase (enzyme) and oxygen are reacted in the presence of magnesium ion. Luciferin and luciferase are the same cofactor and enzyme present in fireflies that yields their namesake light. The products of the reaction are AMP (adenosine monophosphate), inorganic phosphate, carbon dioxide, and light (photons). The reaction, just as in fireflies, produces light. This light can be quantified and used to correlate to an amount of ATP. However, the amount of ATP does not necessarily relate directly to the number of microorganisms or bacterial cells or colonies. In addition ATP may be from non-microbe biomass such as beef blood whereby the amount of ATP would not be related to microorganisms.
The lack of correlation may be due to the variation in ATP concentration within cells and the degradation of ATP in dead cells. ATP is found in all living cells, but the amount of ATP in cells can vary significantly. For example, procaryotic cells have about one hundredth the amount of ATP as eukaryotic cells and different strains of bacteria will contain significantly different amount of ATP. In addition, if a cell is growing or about to divide, it will contain more ATP than a dormant cell. Furthermore, cells that have just died contain ATP and even dead cells may contain ATP. In dead cells, any ATP present may degrade, often caused by a reaction between ATP and intracellular enzymes contained within the dead cells. All of these variables in ATP concentration means that ATP testing is limited as a means to quantify the number of microorganisms or bacterial cells or colonies. However, ATP testing remains a method to qualitatively determine the presence of biomass including microorganisms.
Thus, the detection of ATP can be used to determine the presence of biomass, whether viable or nonviable. The ability to detect nonviable biomass is important, for instance, in testing a surface for cleanliness, because nonviable biomass (dead cells) such as food residue, can provide a medium for living cells to grow.
Typically, the luciferase, luciferin, and magnesium ion are sold as a single combined reagent, not as individual reagents. The luciferase must be at the proper pH of 7.8 in order to be effective, usually achieved by employment of a buffer solution. If the proper pH is not maintained, the reaction will not work efficiently, and the results will be erroneous. However, luciferase is unstable while in solution, and will degrade, particularly at higher temperatures. Generally, at room temperature, the luciferase solution will remain effective for a period of hours whereas at near freezing temperatures, the luciferase solution will last for a period of days. In addition, luciferin in solution is light sensitive. Light causes the dissolved luciferin to degrade. Once the luciferin has degraded, no cofactor remains to unleased the bioluminescent reaction resulting in false negatives.
To prevent degradation, the luciferin and luciferase can be dried and protected from light. Methods for drying include, but are not limited to, freeze drying and lyophilization. The luciferase is more stable if kept out of solution. When ready to use, the dried luciferin and luciferase are dissolved in, for example, water containing an appropriate buffer to form an aqueous solution having the proper pH.
By mixing the luciferase/luciferin reagent with a sample taken from a test surface, extracellular ATP is immediately reacted and detected. However, intracellular ATP cannot be detected unless the ATP is first extracted from within the cells. Typically, this is accomplished by mixing the sample with an extraction reagent (releasing reagent) which extracts the ATP from within the cells. The extracted ATP then can be mixed with the luciferase/luciferin reagent to produce the observable reaction. It is important that the extraction reagent chosen does not inactivate the luciferase/luciferin reagent.
The luciferase/luciferin reagent cannot be stored with the extraction reagent as it will inactivate the luciferase and/or the luciferin over time. It either is inactivated, no light will be produced when combined the ATP. Therefore, the luciferase/luciferin reagent and extraction reagent must be stored separately until the time the test is conducted.
The bioluminescent reaction of ATP and luciferase/luciferin has traditionally been conducted using two basic types of systems: vial systems and all-in-one swab devices. A vial system uses a series of vials containing the reagents necessary to conduct the ATP tests. An all-in-one swab device provides all of the reagents and the swab in a self-contained apparatus.
In a vial system, for example, a first vial contains the extraction reagent, a second vial contains dried luciferase/luciferin reagent, and a third vial contains a buffered solvent to rehydrate the luciferase/luciferin reagent. At the time of the test, the solvent is added to the vial containing luciferase/luciferin.
A sample is collected by wiping a prewetted swab across the testing surface. Typically, the swab is pre-wetted with saline. The swab containing the sample is placed in a test tube. Next, the proper amount of extraction reagent from the first vial is pipetted into the test tube containing the swab. After sufficient time has passed to ensure ATP extraction, the buffered solution containing hydrated luciferase/luciferin reagent is pipetted into the test tube and the luciferase is allowed to react with the ATP. The test tube is then placed into a luminometer where the amount of light produced by the reaction is measured. If more than one sample is taken, each sample is placed in its own test tube.
While vial systems produce correct results, there are deficiencies. One large problem is that the quantity of luciferase/luciferin solution prepared must be used within a short time period. If leftover solution is saved for later tests, the luciferase will degrade and ultimately become ineffective thus producing no reaction even in the presence of ATP. This problem is compounded by commercial producers of the luciferase/luciferin reagent that only sell the reagent in quantities that produce an amount of solution that is greater than that needed for individual tests. Furthermore, the dried enzyme is relatively costly. Thus the vial system results in waste of expensive reagents when only an individual test is required.
Another shortcoming of vial systems is that accurate pipetting and mixing of reagents is required. A pipette is used to transfer the reagents from vial to vial or vial to tube. While pipetting is accurate, it is laborious and time consuming. Further if any of the vials or pipettes are not sterile, any biomass contaminant will produce a false positive for the presence of ATP.
The all-in-one swab devices apply the same reaction as the vial systems but keep all of the reagents and swab in a self-contained apparatus that fits into a luminometer. All-in-one devices typically contain a swab that is placed in a plastic tube containing several chambers. An advantage to this system is that a unit dose of each reagent is provided for one test, thus avoiding waste of reagents when only one test is required. However, a certain procedure must be followed using an all-in-one device to ensure that the reagents are combined at the appropriate times.
In a typical all-in-one device, a swab pre-wetted with a wetting solution is placed in a sealed tube until ready for use. The wetting solution may contain an extractant. The sealed tube prevents evaporation of the wetting solution. At the appropriate time, the device is opened, the pre-wetted swab is removed, and a sample is collected by wiping the swab along the testing surface. If present, an extractant will extract intracellular ATP from the sample collected on the swab. The swab is then placed back in the tube and the tube is resealed and ready for ATP reaction with the luciferase/luciferin reagents.
Dried luciferase/luciferin reagents are kept in a dry, stable state in the tube until mixed with a buffer solution. The luciferase/luciferin may be kept isolated from the wet swab by placing the luciferase/luciferin in a separate chamber in the tube which can be broken to expose the luciferase/luciferin to the buffer solution. Alternatively, the luciferase/luciferin may be in the form of a pellet that can be placed in a sealed container or can be stuck to the bottom of the tube.
A sealed chamber at one end of the tube contains the buffer solution. The tube is squeezed to break the barrier wall between the chamber and portion of the tube containing the swab, resulting in release of the buffer solution. The tip of the tube is then shaken to allow the luciferase/luciferin regents to mix with the buffer solution, hydrate, and mix with the sample on the swab. The entire tube is then placed in a luminometer where the amount of light produced is measured.
While the all-in-one systems have overcome many of the problems of the vial systems, they have other shortcomings. For example, all-in-one systems are costly to manufacture since a complex tube arrangement is needed that is resealable and contains a breakable chamber to hold the buffer solution and possibly a second breakable chamber to hold the luciferase/luciferin.
Whatever system is used, the swabbing of the test surface should not itself contaminate the test surface. Thus, for example, the extracting agent used on the swab should not contain toxic chemicals that will leave toxic residues on the test surface.
Again, whatever system is used, the resulting tube containing the luciferase/luciferin and ATP is placed in a luminometer to read the light produced during the reaction. In the past, luminometers were designed with detectors aimed perpendicular to the axis of the sample tube so that when the sample is inserted in the luminometer's measurement chamber, the detector views the light produced by one side of the sample. Side-viewing detectors are appropriate if ATP is measured in solution. However, such detectors can be a problem in swab systems if the sample is located on only one side of the swab, and that side is placed on the opposite side of the detector, then the amount of light reaching the detector will be less. Thus, the quantitative light measurement becomes dependent upon how the sample is placed in the luminometer.
More recently, a luminometer with a bottom-reading detector was developed which avoids the problems of side-viewing luminometers. A bottom-reading luminometer views the bottom of the sample tube and provides an accurate reading independent of the orientation of the sample and whether the sample is in solution or absorbed onto a swab.
In co-pending application entitled Method and Apparatus For Rapid Hygiene Testing filed concurrently herewith, a method and device for bioluminescent ATP detection is described which provides a sampling device having an absorbent tip, such as a swab, wherein luciferase/luciferin reagent is immobilized in the tip. The luciferase/luciferin reagent is applied to the tip, or the tip is dipped into the reagent, and then the tip is dried by, for example, refrigerating at four degrees Celsius overnight, freeze drying, or lyophilization. The tip may be made out of a sterile fiber such as cotton or a synthetic fiber such as DACRON.
Several drops of a suitable wetting or extraction solution, such as chlorhexidine diacetate (CDA), water, or surfactants, are added to the test surface. The tip of the sampling device is wiped across the solution which activates the luciferase/luciferin reagent in the tip. The sampling device is then placed in a counting tube which in turn is inserted into a luminometer. By measuring the amount of light produced by the reaction, an amount of ATP can be determined. The sampling device containing a luciferase/luciferin enriched tip provides advantages over all-in-one systems since it is not as complicated nor as expensive.
Often it is desired to determine if and how quickly bacteria are growing. The all-in-one device or the sampling device above give a single reading and this they do not provide information as to whether the bacteria are growing.
It is the object of the present invention to provide sequential measurements of liquid culture samples to determine the presence and growth of bacteria through the detection and measurement of ATP over time. Another object is to use unit dose dried in the bottom of a counting tube and luciferase/luciferin dried in the swab tip. A further object is to use CDA dried in the bottom of a tube and luciferase/luciferin dried in the bottom of a counting tube. A further object of the present invention is to avoid waste of expensive reagents. Another object is to provide a system that uses a bottom-viewing detector and a non-light-absorbent tube that can be placed directly in the luminometer. Finally, an object of the present invention is to perform the test without the need to transfer reagents to a different container before being placed into the luminometer.